Summary Studies and research on bee products with a view to their superior utilization CORNELIA (DOŞTEŢAN) ABĂLARU “Lucian Blaga” University in Sibiu The Faculty of Agricultural Sciences, Food Industry and Environmental Protection The fundamental field of PhD: Engineering Science The field of PhD: Industrial Engineering The field of documentation: Beekeeping and the products obtained from this activity STUDIES AND RESEARCH ON BEE PRODUCTS WITH A VIEW TO THEIR SUPERIOR UTILIZATION Utilization in the shape of bee cocktails, functional food and cosmetic products on the basis of bee venom SUMMARY Scientific guide: PhD student: PhD Professor Engineer: Engineer: Cornelia Carmen Ioan DANCIU ABĂLARU (DOŞTEŢAN)

Sibiu November 2014

Summary Studies and research on bee products with a view to their superior utilization

TABLE OF CONTENTS Introduction page 4 Aim and objectives of research page 5

THE FIRST PART THE CURRENT STAGE OF RESEARCH

THE BIBLIOGRAPHICAL STUDY OF BEE PRODUCTS Chapter 1 Bee products as raw materials page 7 1.1 Generalities (definition, classification, composition, production) 1.1.1 Honey 1.1.2 Pollen 1.1.3 Bee bread 1.1.4 Propolis 1.1.5 Royal jelly 1.1.6 Drone larvae 1.1.7 Bee venom 1.2 Harvesting, packing and depositing methods of bee products Chapter 2 page 51 Bee products and health 2.1 Therapeutical utilizations and actions 2.2 The necessity of consuming food supplements (bee cocktail), functional food and the use of cosmetic products on the basis of bee products

THE SECOND PART PERSONAL STUDIES AND RESEARCH ON BEE PRODUCTS WITH

THERAPEUTICAL USE

BEE COCKTAILS, FUNCTIONAL FOOD, COSMETICS Chapter 3 Personal research on the quality of bee products – raw material page 70 3.1 Materials and methods 3.2 Results and discussions Chapter 4 Bee cocktails ( registered trademark OSIM). Page 89 4.1 Processing raw materials and getting bee cocktails in the viewpoint of ISO 22000:2005 Chapter 5 Functional food page 106 5.1 Functional food “APILIFE” Chapter 6 Products with bee venom page 121 6.1 Revitalizing gel with bee venom, hot pepper, mint and pine 6.2 Regenerating and nourishing face gel with bee venom and royal jelly 6.3 Solution of bee venom – spray

Chapter 7 Conclusions and perspectives page 134 7.1 General conclusions 7.2 Recommendations 7.3 Personal contributions and future tendencies of research development Bibliography page 137 Annexes page 142 The list of notations and used symbols page 172 The list of figures page 173 The list of tables page 175 Curriculum vitae page 176 The list of published papers page 178 THE STRUCTURE OF THE THESIS The PhD thesis was worked out on the basis of research done throughout several years in my own apiary, then within the Apilife company and the collaborations with The Romanian Society of Beetherapy, with the Hofigal Society, with The National Institute of Research – Development for Cryogenics and Isotope Technologies in Râmnicu Vâlcea, with USAMV in Cluj, etc. The paper is divided into seven chapters, conclusions, bibliography and annexes. It has 180 pages out of which the reference part represents 65 pages. The master’s degree paper contains 32 figures and 16 tables, and the annexes are rendered in 28 pages. In order to

elaborate the thesis, there were used 100 bibliographical references. The dissemination of results is represented by the list of papers presented in national and international beetherapy Congresses and Conferences. The PhD thesis is divided into two different parts:

I) The reference study II) The experimental part

In the reference part, divided into two chapters (1 and 2), I deal with data in

the specific literature: the definition, classification, physical-chemical composition, production, harvesting methods, packing and depositing of bee products analysed throughout this paper, namely: honey, raw pollen, maiden wax, propolis, royal jelly, drone larvae and bee venom. In the experimental part, divided into four chapters (3, 4, 5 and 6) there is personal research concerning bee products with therapeutical use: personal research concerning bee products as raw material (chapter 3), bee cocktails (chapter 4), functional food with the products of the beehive (chapter 5) and cosmetic products with bee venom (chapter 6). The paper ends with some conclusions and perspectives (chapter 7), bibliography, annexes, the list of notations and used symbols, the list of figures, the list of tables, my Curriculum vitae and the list of published or presented papers. Before moving on to the presentation of the paper, I ought to thank and express my gratitude to those who supported me in elaborating and completing my master’s degree thesis. First of all, I want to thank God for everything I am. I also want to thank PhD Prof. Eng.Vasile Jâşcanu, the scientific guide of the thesis, for his guidance and competence in coordinating my activity throughout the elaboration of the thesis. I also want to thank PhD Prof. Eng. Ioan Danciu for his time and support for completing and presenting the thesis. Special thanks to Mrs. PhD. Pfarm. Gabriela Vlăsceanu for understanding, support and especially for the material and logistic support at my disposal, to Doctor Ştefan Stângaciu, to Mrs. Iuliana Crişan, … I thank the board of examiners for assessing and presenting the master’s degree thesis, for the honour of analyzing my thesis. Last but not least, I want and need to thank my family: my mother for supporting me, my husband, our 11 – old month son, my dear friend Romeo for his support and understanding throughout the elaboration and completion of the master’s degree thesis, for his moral support, for his understanding and unconditioned support throughout the research period.

I dedicate my success to them….

The aim and objectives of research The aim of this project on “Studies and research on bee products. Their utilization as bee cocktails, functional food and cosmetic products on the basis of bee venom” is the documentation, experimentation and estimation of some technological alternatives of getting some complex food and cosmetic products based on products of beehive, in view of the optimization of the:

chemical composition (by association) way of preserving effect on health The research activity within the project aims to practically get three categories

of bee – fit therapeutical products: bee cocktails functional food cosmetic products

and the results are stipulated to be of interest, both on the national and the international market, in the field of food supplies and cosmetic products based on products of the beehive. Taking into account the importance of chemical compounds in bee products in keeping and improving health, as well as the curative characteristics of fruit and medicinal plants (analysed again and valorized with the help of modern technologies), we have in view to get some “bee-fit” therapeutically efficient products at the level of the human body. Thus, the research aims the following:

I. The products of the beehive were studied in the first stage Honey Pollen Bee bread Propolis Royal jelly Drone larvae Bee venom

considering the chemical composition, in certain compounds with benefits on the human body.

II. The second stage aimed to get some bee-phytotherapeutical products such as “bee cocktail”; and it had in view both the finding of some technological alternatives of production, and the discovery of the best ways of conservation and dosage.

III. The third stage was concerned with the study of the possibility of getting some functional food based on bee products, associated with fruit and medicinal plants, in order to keep and improve health.

IV. The fourth stage aimed to formulate and test a range of cosmetic products based on bee venom, simple or associated with medicinal plants.

Chapter

1.1.1 HoA

in sugasecretio

T

“kiss” athroughmomenteliminatand end

T

processi( sacchacompletdilutionThe purprocess,space.

Aquartes centrifu

r 1 1.1 Bee prooney According t

ar and vitamons of trees a

The nectar among beesh which smat when it ited. The pr

ds with putti

The procesing. The enarose, maltotely assimil

n of nectar rpose of con, the activity

At the momlidded”), b

uge them and

oducts, raw

to the dictiomins, madeafter having

that bees s (the kiss oall drops ofis enrichedroduction ofing lids on t

ssing is acnzyme proceose, etc.) inate them duor manna wncentration y of microo

ment of comeekeepers td pack hone

materials –

onary, honeye by bees g been prick

suck from fof bees) whif nectar are

d with the f honey by the cells of t

chieved thressing comento simple, uring winterwith saliva

is to reducorganisms (

mplete mattake the framey in big dis

– general co

y is a semilfrom the n

ked by aphid

flowers is tuich particip

e transferredenzymes thbees starts

the honeyco

rough two es out of theassimilablering. The hy(the vector

ce the waterthe osmotic

turation of mes out, pushes.

oncepts

iquid substanectar of fdae.

urned into ate in the pd from one hat ennoble with harveomb where

ways: enze necessity e sugars (gluydric procesr of sugary r content inc pressure)

honey by but them in a

ance, sweetflowers, bu

honey throprocess of m

bee to anoe it, then thesting the nhoney was

zyme procof bees to tuucose and fssing is achi

enzymes) n order to oand the dec

bees in the a clean room

t and flavouut also from

ugh some smaturation oother, this bhe water e

nectar or thestored.

cessing andurn complefruitose) in ieved in twothen conce

obstruct the crease of the

beehive (“m , take the

ured, rich m sweet

sort of a of honey being the excess is e manna

d hydric ex sugars

order to o stages: ntration. enzyme

e storage

“of three lids off,

H

lids, etcT

specificof honehoney) temperait was idensitygot the maximuchemicawhich hfrom onrecognizfact thatplants. honey whumiditpigmentglucose substanccomes fin a very(vitaminpyrodixgluconiccaroteneone to honey iand glucthe phoenzymemanna hwhich pproducethose acompossubstancnoninteninvertasthe reac

Honey can c, by filtratioThe flavou

c flavour of ey is very to dark bro

ature (20°C)inadequately

y of honey isdensity of

um 5 for theal reaction ihelp to certine range to zing the orit the morphThe polliniwithout a wty. Honey ts, acid and (31%), sacce) (specififrom plants.y small quan B2), Nico

xin (vitaminc, citric, mae, chlorophywhom the s due. The cose), the d

osphatase. Tes in nectar)honey have produce theed by the madded throusition and qces, espensification se, the amylctions of tra

be cleaned on or cleani

ur of honeylinden tree,sweet. The

own (manna). If humidiy obtained s strongly inf 1,4710. Te manna oneis a stronglify the authanother wi

igin of honehology of thic spectrumwell-closed consists ofaromatic co

ccharose (1ic to manna. The nitrouantity, belowol acid (vitan B6). Thalic, chihlimyll and the presence oenzymes in

diastasis (it cThey have ) and animaa much hig

e manna, thmushrooms ugh the saquality is mcially theof enzyme

lase, the catansforming s

by possibleing the abov

y is given by, raspberry be colour ofa honey). Gity is higheror kept, annfluenced bThe aciditye. The normly acid one.henticity of ith floral hoey and the

he pollen grm of honey

lid absorbf: water, caompounds. 1%), maltosa honey). Wus substan

w 1%. The eamin B3), ve aromati

mbar, formiderivatives

of some smn honey areconverts staa double

al (the enzymgher origin (hose secreteand alga waliva of bmostly giveermolabile. es. Amongtalase. The astarch into d

e foreign bove layer whiy the sourcbush, etc. uf honey ma

Good honeyr than 21%,

nd it will deby its conteny of honey mal values o

The electrmanna hon

oney. The pcorrect appanules is abis the loyas the smellarbohydrateCarbohyd

se and otheWater is bet

ces are exprexisting vitaitamin K, foc compounc, acetic, bu of chlorop

mall quantitie: the inverarch into dexorigin in thmes in the (the enzymeed by insec

which contambees). The en by the c

High tg the existamylase (thdextrines an

odies: smallich appears e-plant of n

up to bitter (ay differ fr

y (16-18% h, honey flowegrade rapidnt of water.

is maximuof pH of honric conduct

ney. The elepollinic spepreciation ofbsolutely chal mirror ofl around, aes, pollen, rates (sugaer disacchartween 16 –ressed in pramins: thia

folic acid (vnds terpenutyric, lactihyll, xanthoies of vitamrtase ( it conxtrines), thehe flower saliva of smes in the sap

cts during thminate the characteris

content of temperatureting enzymhe diastasis) nd further a

l wings, beeafter a shor

nectar, from(the chestnurom almosthumidity) isws like watedly, being oHoney withum 4 for fney are betwtivity is onectric conduectrum is thf the type o

haracteristicf its origin.and in a hu

minerals, ears or glucidrides (7%),

– 23% and irotein equivamine (vitamvitamin M), nes, aldehydc amino-acophyll. Themins, aminonverts sacche glucose-oxhoney: ve

mall bees). Tp of plants the process drops of mstics of hoenzymes. E

e causes mes in hone

is the enzyand it is an e

es, larva, smrt period of

m discret (acut honey). Tt colourlesss viscous ater which meof low qualh 20% humiflowers honween 3,5-4,e of the par

uctivity diffhe main cri

of honey, du for each sp. Inadequatumid are it enzymes, v

des): fruitose, melezitoseit is “biolog

valent and amin B1), rib

biotin (vitades, esters.ids. The pi

e pollen (5%o-acids, proharose intoxidase, the egetal (the The enzymethat feed theof digestio

manna on ploney, its cEnzymes ar

the irreey I ment

yme which cenzymatic i

mall wax f time. cacia) to

The taste s (acacia t normal eans that lity. The idity has ney and 5, so the rameters fers a lot iteria for ue to the pecies of tely kept

absorbs vitamins, e (38%), e (sweet gical”, it

are found boflavine amin H), . Acids: igments: %) is the oteins in fruitose catalase, existing

es in the e insects

on, those ants and chemical re labile eversible ion: the catalyses indicator

of honereactionhoney hmaking existingphosphahigh in and thisprolongresponscomposnamely

1.1.2 PO Awith flo Fsource oetc. For Iand som

ey being thens of decomhelps to con

evident ofg in honeyates, siliciumthe manna

s is due to ged contact ible for the

sition and ththeir active

OLLEN According t

owers, whichFor bees, pof substancer human beinIn order to

me data are n

e most resimposition ofnfirm the typf installing y are: potm. The minhoney. Ththe inadeqof honey w

e extraordinherapeuticale principles.

to the dictioh represent ollen is thees indispenngs, pollen understand necessary.

isting enzymf peroxides pe of manna

the fermentassium, soneral substanere may be

quate extracwith nonfo

nary qualitiel effects of .

onary, the pthe male se

eir main sousable to theis a source the origin

ww

me. The cawith oxyge

a honey, thentating proodium, calnces in hone situations ction and stod metallices of honeyhoney are

pollen is a pexual cells.urce of foodeir life: lipidof vitaminsof pollen an

ww.biologie.

atalase is anen release. Te confirmatiocess in a cium, magey have a lowhere the

torage of hc surfaces (y, so highlyestablished

powder orig

d, the uniqudes, minerals, lipides, prnd the way

uni-hambur

n enzyme wThe researcion of thermchemical wgnesium, cow content maximum l

honey (in du(wraps). Th

y appreciated by the pla

ginating in t

ue source ofl elements, roteins, enzybees pick u

rg.de

which catalch of the camic degradaway. The mchlorine, su

in floral holimits are eust), in thehese substaned by all of ants visited

the stamen o

f proteins, tvitamins, eymes, mineup pollen, a

lyses the atalase in ation, the minerals ulphates, oney, but exceeded case of nces are

f us. The by bees,

of plants

the main enzymes, erals. an image

Tare: thewhich iovary inthe stigm IThroughstigma aplace wthe fema other p(entomoto one s PThere athe flow

Ia. The pthe hardto help the botsb. Durinc. Aftermediumd. Behinthe cell

The role of ovary, thes the male n the shape ma has a stiIn order to h germinatiand the styl

when the nucale germinaSome plantplants prodophilous pospecies of plPollen harv

are some bowers. The be

In the procepollen cells d bots of thepacking the

s on the medng the flightr having go

m feet, in an nd it, there with its hea

f a flower is style and tsexual cellof a colum

icky surfacegive birth ton, the pollle reaching clei in the mal cell. ts produce aduce a heollen). 80% lants in its fvesting by b

ots on the boee moves fa

ess of harveon the head

e front feet.e pollen. Thdium pair oft, the bee paot in the beempty cell comes ano

ad.

to producethe stigma. . The ovary

mn. At the te to which thto the seed,len gives bithe ovary a

male germin

a light polleeavier, stickof pollinati

flight, avoidbees ody of bees

ast among st

esting the pod, appendix. The bots ahe hinder paf feet. The aacks the poleehive, the or partly fither bee wh

e seeds. TheThe male

y becomes aop of the sthe pollen gr the pollen irth to a tubachieving fenal cell at th

n, this is trakier pollenion is achievding interspe

s. The polletamens and

ollen grainsx and the firare moistenart and the abdomen is llen and stufbee downl

lled. hich crushe

e essential fpart is the a fruit. Thetyle there israins adherehas to reac

be which paecundation. e top of the

ansported bn, and thived by melecific visits

en attaches tthe pollen a

, bees go thrst segmentsed with neclast segmencleaned wiffs it in the loads the sm

s and press

female partsstamen. Th

e style is a s the stigmae where theych the stigmsses througIn the ovar

e pollinic tub

y wind (aneis is translliferous bees (Ialomiţean

to the bots attaches to t

hrough severs of the chectar or with nts of the cth the bots osmall baskemall basket

es the polle

s of a typicahis producesprolongatioa. With moy germinate

ma from the h the interio

ry fecundatibe join the n

emophiloussported by es. It confinnu 1987).

when the bthe bots.

ral stages: est are “com regurgitate

chest are cleon the hindeets on the hits with the

en on the bo

al flower s pollen, on of the st plants e. anthers.

or of the ion takes nuclei in

pollen), insects

nes itself

bee visits

mbed” by ed honey eaned by er feet. inder help of

ottom of

The polchanges Pollen hThe polentrancethrough Ttimes a

The orgWhen ppollen dependiyellow with facpleasanthumidithe devdried). The sizplant, thpollen gthe custespecialThe phhumiditsubstanc 1.1.3 Be

llen thus des biochemic

harvesting llen harveste, through

h which beesThus the poday in the w

ganoleptic pollen is har

varies froming on the fwith the sncelie, light tly mellowity of pollenvelopment o

ze and the he duration grains counttomer’s eyelly because ysical and ty of pollences 1-7 %.

ee bread

eposited is ccally turning

by the beeting by the a series ofs can pass oollen falls inwrap, then p

characterisrvested, it l

m white to flowers visinap dragon,grey with e, with an in at harvestof microorg

weight of pof harvest at a lot on a ces. The sizeit is recommchemical c

n is of 15-20

covered witg into bee br

keeper beekeeper t

f devices caonly after hanto a collecputs it into t

stics of polllooks like sblack, the p

ited by bees, yellow lemelm trees, aintensely siting is veryganisms, th

pollen graiand the distcommerciale of the gramended to bcharacterist0 %, protein

th a layer oread.

takes place alled “polleaving downcting box whthe freezer o

len small grainsprevalent os, for exampmon with rand almost imilar perfu

y high, that hat’s why it

ins may diftance to the l point of vi

ains does nobe consumetics of pollens 7-35 %,

of honey, b

at the entraen collectornloaded the shich the beor in the pol

s with an irone being yple: light yerapeseed, grblack with ume to theis why it ist must be

ffer a lot desource flow

iew. Crusheot count on ed in a mixtuen. At the minvert suga

eing to go

ance in the rs” whose psmall bags wekeeper emllen drying

rregular outyellow. The ellow with hreenish withpoppy. Th

e flowers its a very gorapidly pro

epending onwer. The sized pollen do

a therapeuure. moment of har 1-48 %, f

through con

beehive, atprinciple iswith pollen

mpties twice devices.

line. The cocolour var

hazelnut treh maple tree taste of p

t comes frood environm

ocessed ( fr

n the specieze and the woes not lookutical point

harvest, thefat 1- 18%,

ntinuous

t the bee s a grate . or three

olour of ries a lot ees, dark ees, blue pollen is om. The ment for rozen or

es of the weight of k good to of view,

e level of mineral

Bbring polhoney anpollen gradded bythrough aafter a pe Ddifferent

thwth

thlaex

MaideffervescThe cheacids anddue to itsincreasedand antibdestroyedwax is th200-875 M 1.1.4 Pro

Pand the beehive f Pblack, hasoluble in120 °C. A– marie,

The contThe cont

Bee bread, orllen in the bend enzymes arains , then ty bees, of mia series of beriod of natu

Due to the ffrom the ini

he first stagewhich will cohese bacteriahe second actobacillus,xchange, its

den wax is dacence. mical compd lactic acids higher cond acidity, whbiotic valued, leading tohe followingmg/kg; vitam

Maiden wax c

opolis

ropolis is a bark of treefor several propolis look

aving a flavon water. Its sAt 37 °C it sit splits up ina viscous paa liquid parttent of wax tent of impu

r “the bread eehive, depoand other nothey put a pricroorganismbiochemical ural effervescfact that theitial pollen. Te consists of onsume the a by selfsuffostage is p which usconcentratio

ark brown, it

position: Mad for the foodtent of simphich makes e is three tio an easier ag: carbohydrmin E 1.7 g/contains mo

resinous maes. Its phys

purposes. ks like a sticoured smell specific weigsoftens, and nto two diffeart which got (propolis wvaries betwe

urities varie

of bees” maosit it in the ourishing subrotective layms, of high te

changes ancence. e changes oTurning of p

f the developwhole quantocation. produced byses glucideson reaches 3t tastes swee

aiden wax id of bees. It

ple sugars, viit easily ass

imes higher.assimilation rates (glucid/kg; vitamin re vitamin K

aterial with tical charact

cky mass ofof resin andght is of 1.1at low temperent parts:es to the bot

wax) which feen 7,5 and s between 18

ay be the mocells, press

bstances fromyer of honey.emperature ad structural

of pollen arpollen into mpment of a btity of oxyg

y the deves as the sou.2%; the con

et-bitter-sour

is rich in cat is a naturalitamin K, ensimilable. A. The externby the body

des 35%); lipC 6-200 mg

K and folic a

the firmness eristics and

f different cd balm. It is 12 – 1.136 geratures it is

ttom floats on the 35 % (Ivano8 and 34% (

ost valuable it with their m the salivar. Here, undeand humiditmodificatio

re biochemicmaiden wax i

acterium calgen, and fina

elopment ource of oxygncentration orish due to th

arbohydratesl product, m

nzymes and s compared nal cover oy. The chempides (1-6%)g/100 g of pracid than the

of wax harvcolour diff

colours, rangsoluble in a

g/cm³. The ms breakable.

surface of wov, 1981) (Makashvili,

product of tsmall head,

ry glands useer the influenty in the beeons, turning

cal, the proinvolves sevlled pseudomally this lead

f an anaergen, produciof vitamins Bhe process o

s, proteins, vmore valuabl

amino-acidsto the polle

of pollen, thmical compo); caretonoidroduct. bee pollen.

vested by befer and it is

ging from galcohol and emelting poinIf it is easily

water

, 1972)

the beehive. , mix it withed as a bindence of substaehive, polleninto maiden

oduct obtainveral stages:monas , aerods to the dea

robic bacteing lactic acB also increa

of

vitamins, amle than the ps, as well as en, its nourihe exine, isosition of mds (provitam

ees from the s used insid

green, brownether and he

nt is betweeny warmed in

Bees h little er for ances

n goes n wax

ned is

obian, ath of

rium, cid in ases.

mino-pollen

to its shing

s also aiden

min A

buds de the

n and eavily n 70 – n bain

The coloyellowishbrown, ebrown. Tpoplars, flavouredto the prohas a verpropolis propolis Marchencompositoil, iron, are addedacid (actbalms, faluminiuantibioticto the veg

5 3 1

The prodspecies oconiferouprocessed Tis achievtime whebeehive, 1.1.5 Roy

Rtheir 6th athree stag Rlittle soudifferent colony omineral sabout 2/3fractionsconcentraproductio

our. It varieh-greenish, yetc. For instThe smell. wax, honey

d resin whicopolis in othry powerfulhas a bitterare pungen

nay, 1976). tion. Propolmicroeleme

d. It representive against ferments, mum, vanadiuc substancesgetal species5 % resin an0 % types of0 % essentiaduction by b

of trees, espeus trees and d by speciali

The productioved bt speciaen it turns inaccording to

yal jelly

Royal jelly isand 14th dayges of larval

Royal jelly isur and astrin

species or cof bees.The salt (Rainer 3 of fresh ro. A very imation of the on of royal je

s according yellowish-brance, the prThe Europe

y and vanillch becomes vher areas andl smell of cir and pricklnt. The flavpH varies

lis is made oents, to whicnts a mixtureGram posit

microelementm, wolfram, resin, flavos from whichnd balms, f wax al oil, quantibees. Bees cecially on thpoplars, Salized bees byon of propolalized bees, nto plastic. Oo the region.

s a substancy of life, sub

developmens an apparengent. It usuacolonies of bmain compKrell, “Valu

oyal jelly, bmportant sub

acid is a goelly, this mu

to the geogrown, brownrevalent coloean propolisla. When it volatile. Thed regions of innamon. Thling taste (M

vonoides in between 5.

of vegetal rech pollen, fle of substanctive and Grats (silicium,

m, iron, goldoured acids, h it was harv

ties which acollect a flave poplar andicaceae and

y mixing themlis takes placon warm da

One can harv

e secreted bstance whichnt. ntly white crally has a vebees. Its stabponents of rue added pr

but in the drbstance, exisood indicatoust be higher

graphical aren reddish, daour of the B

s usually smis burnt, it

e smell of thRussia (Ma

he taste. SoMakashvili,

propolis ta.2 and 5.7 esin, balm olavonoides, sces, especialam negative, magnesiumd, iridium, acids. The c

vested, but, o

are similar tovoured substad alder-tree bplum trees. m with salivce at the samays, when th

vest a quantit

by the hipofah will feed t

reamy and very stable cobility may broyal jelly aroducts fromried part, prosting in the or to check tr than 1.8% o

ea and the park brick-colBrazilian pro

mells nice ant gives off he Georgian akashvili, 19our and som

1972). Moaste bitter a

(Bracho etof different secretions olly: the flavoe embryos), m, copper, calcium, ca

compositionon an averag

o any kind ofance which lbuds, on theThe resinou

vary secretionme time withhe temperatuty of 100 – 4

aringian glanthe queen an

viscous subsomposition, e the basis o

are: water, pm beekeepingoteins and sroyal jelly,

the quality oof the dried m

plants of origloured, browopolis is rednd mellow a delicate spropolis is

972). The Litmetimes bitte

st of the Band astringet al., 1998)composition

of the salivaronoide deriv

types of wmolybdenu

admium, con of propolisge, it contain

f propolis. looks like re

e leaves, budus materials tns and wax.

h the producture is higher400 grams o

nds of the ynd the saplin

stance. Its taeven the on

of the genetiproteins, sugg , FAO, 19sugars are b, is the 10 Dof royal jellmaterial.

gin: light yewn greenish,d, green or like the budsmell due togenerally sithuanian proer. The Georazilian typ

ent (Jeanson). The chemns, wax, essery glands ofatives, the fe

wax, amino-aum, arsenic,obalt, stront varies acco

ns

esin on at leads and the bathus collecte

tion of honeyr than 20°C

of propolis fr

youngest beeng during the

aste is specine obtained ic stability ogars, lipides

996). Water.by far the hiDHA acid. ly. In the or

ellow, , dark clear ds of o the imilar opolis orgian es of

n and mical ential f bees erulic acids, , tin, tium), ording

ast 20 ark of ed are

y and C, at a rom a

es, on e first

ific, a from

of the s and . It is ighest The ganic

1.1.6 Dro Drone laday of larepresentthen trituDrone larIlieşiu. YIlieşiu. It

Tworking is the onworking T(fecundatconditionbee famil Tthe queen(4,6 – 5spermentbigger (6bottom othe matu16 chrom Flarva, 13 Inlarval stathe cell. Tthis day, one, and nympha tweight oof 240 mbecomes Inrecommewith the the larvalThe orgawith fine

one larvae

rvae is whatarval stage, ting 3%, the

urated, homorvae is an 1

Yes! The dist is obtained

There are thrbees and the

ne that ensubees or quee

The differented or not), ns (the plently, the system

The eggs laidn lays the eg5,1 mm), attom, a sperm6,4 – 6,6 mof the cell. Are ovule, 16

mosomes whor a drone, tdays the sta

n order to geage of the droThe best perthe larva rethe wet nursto the one off the egg 0,0

mg after 24 dup to 1500 t

n literature ended one duhelp of a pul covers stayanoleptic che granulation

t is found ana mixture o

e body of thogenized and100% Romanscoverer of by extractin

ree types of e drones. Th

ures the perpens grow, antiation of tthe qualitat

ty existence m of the beed by the quegg. If the cellt the introdmatozoon co

mm), the sperAn unfecund6 from the spich hatch drothe metamorage of nymphet the drone one, namelyriod of collecaches the mse bees laid af adult, in th012 g it reacdays. As comtimes heavietwo methodue to its supump with vay here. haracteristicns. The colou

nd extracted f larval com

he larva drond filtered. nian bee prothis bee pro

ng the whole

individuals he only femapetuation ofnd unfecundathe three cative and quaof the sourc

ehive). een are fecunl in which th

duction of tomes out whrmentom do

dated egg – tpermatozoonones by partrphosis lasts ha. larvae, the b

y the first 10 cting drone l

maximum deva big quantit

he lidded cellches the larvampared to ther. This is cads of collecperior qualitacuum. The

cs of drone ur of drone l

of the cell wmmon food (ne, aged 7 d

oduct. Api= boduct is a g

e content of t

in the beehale capable of the speciesated eggs ouastes is estantitative difces of nectar

ndated or nohe queen laysthe abdomenhich fecundaoes not openthe diploid en), the unfecthenogenesis24 days: 3 d

beekeeper is days from la

larvae is on tvelopment, tty of food wl where onlyal stage of 3he weight oaused by the ting drone

ty is the onesecond meth

larvae. Thelarvae is wh

where a futu(pollen, mai

days old and

bee, lar=larvgreat Romanthe cell with

hive that formof laying egs. It lays feut of which dtablished byfferenciated r and pollen

ot according s an egg is thn it is pres

ates the egg,n and an unegg- containcundated eggs. days the stag

especially iaying the egthe 10th day the weight is

which helps thy air and hum380 mg on thof the egg la

feeding regilarvae are d

e by extractihod is by sq

e aspect is hhite-grey ; th

ure drone deiden wax, hd the larval c

va, n=Nicolanian beekeep

h the help of

m the colonggs is, normacundated eg

drones grow.y: the struc

food and thin nature, th

to the size he normal onssed and it, if the cell nfecundated ns 32 chromg – the haplo

ge of egg, 8

interested in gg, before the

after havings a little belohe larva ovemidity pass. he 11th day, aid on the firime. described. Tion of the coqueezing the

homogeneoue firmness:

evelops on thoney and wcovers whic

ae (Nicholasper, Mr. Nia pump.

ny: the queenally, the queggs out of w. cture of thehe environmhe strength o

of the cell wne of honeyct also openof honeycomegg falls o

osomes (16 oid egg- con

days the sta

the period oe bees put lig laid the eggow the maxiertake the staStarting fromand then an rst day, the

The first andontent of the

e honeycomb

us, a little vismilky, unct

he 7th water), ch are

s), il= icolae

n, the een. It which

e egg mental of the

where comb s the mb is n the from

ntains

age of

of the ds on g. On imum age of m the adult larva

d the e cell b, but

scous tuous,

slightly vtaste : cr Twater 65glucides 1,2%. 1.1.7 Bee“The liqubananas. indicatinthe driedthe acid losing bevenom ca Tcarbohydbiggest qhyaluronprevents Other suinflammanoradren Tvenom bdefence. establishthey manbrought ubrought uup in sum

Chapter

2.1 Ther Tfeatures. – featurefeatures – Tenergeticantibiotic

viscous. Thereamy, easilyThe physical5 – 80 %; th1 – 5,5 %; l

e venom uid bee veno

It is slightlg an acid re

d venom doereaction. Ve

etween 65-70an be collect

The bee vedrates with aquantity 40-5nidaza is a v

the componubstances, suatory answe

naline. The bee venoag and elimiThe secretioed that recennage to get up without pup in spring mmer or autu

r 2

rapeutical uThe origin, co

Knowing thes is highly – pharmacol

The bee honc value, easilc substances

e smell: specy astringent; l and chemihe content oipides 5 – 6,

om is colourlly acid (pH action. The s not look lienom gets d0% of its orted (from onenom is a a powerful p50%, the on

valuable comnent C3 fromuch as hyaluer of the ve

om is a proinated outsidon of venomntly hatched up to 0,15 mpollen, so wwhen the po

umn. Once u

tilizations aomposition ahem is extrem

interesting logical actionney is an exly assimilabl, ferments, v

cific, slightlywithout imp

ical charactof dried sub,3%; ash 1 –

less, with pu5.0 – 5.5); specific wei

ike that, showdried at the iginal weigh

nly one bee)”combinatio

pharmacologne in which mponent enz

m developinguronidaza, phenom. It al

oduct of secde at the mo

m is influencbees do not

mg, and whwithout proteiollen resourcused, the rese

Bee prodand actionsand charactemely importfor biochemns – therapexcellent typle, with real vitamins and

y flavoured purities, parateristics of dbstances 20 – 1,5%; unid

ungent-bitterit changes t

ight is 1.313wing that thenvironmen

ht. When the”. (Michael Son of pepgical action.the quantity

zyme which g and obstruchospholipaslso contains

cretion of thment of pric

ced by the agt have any vehen they are ins, do not pces are rich, erve of veno

ducts and h

eristics of thetant in the th

mists, chemisutic effect is

pe of food wbiostimulat

d mineral ele

( a bit like tasites, fragmdrone larvae– 35%; tota

dentified sub

r taste and flthe colour o3 g/cm³. Howhe volatile contal temperate liquid evapSimics)

ptides, enzy. The compy of venom

splits the hcts the channe A2 and h

s quantities

he working bcking, whichge of bees, enom, only w15-20 days

produce venproduce mo

om does not r

ealth

e main bee pherapeutical sts, biologiss very imporwith a highting features ements.

the smell of ments of larva

e ph 4,8 – 6al proteins 1

bstances 4 -6

lavoured smof blue litmuwever, the wompounds arture in less porates, 0,1

ymes, lipideonent whichis expresse

hyaluronic anels depende

histamine areof dopamin

bees, whichh is, in fact, food and sewhen they a

s old, they gnom. The geore venom threcover and

products leadpractice. Th

sts and researtant for prach nourishing

due to the c

f fresh dregsa.

6,7; the conte10 – 20 %;

6 %; density

mell similar tous paper int

watery solutire responsibthan 20 minmg of pure

es, amino-ah is found id is melitin

acid. The apent on potasse involved in, serotonin

h is stored ia reflex actiason. It has

are 6 days olget 0.30 mgnerations of

han those brothe bee dies

d to their curhe ratio subsarchers, the ctitioners. g, biologicalcontent of na

); the

ent of total 1,1 –

o ripe to red ion in le for nutes, dried

acids, in the . The

pamin sium. in the n and

n the ion of been

d can .Bees f bees ought s.

rative stance

ratio

l and atural

The bee wax helps to make artificial honeycombs and as raw material in the electrotechnical, electronic, optic, cosmetic, pharmaceutical industries, varnishes and dyes, etc. The pollen, propolis, royal jelly, drone larvae and bee venom are extremely appreciated for their therapeutical and biostimulating features. The pharmacological characteristics of bee products are to be found in 10 pages of the thesis. 28 features of honey, 53 features of pollen, 60 features of propolis, 33 features of royal jelly, 17 features of drone larvae, 17 features of propolis were mentioned and justified. 2.2 The necessity of consuming food supplements (bee cocktail), functional food and the use of cosmetic products on the basis of bee products The miraculous features of the mixture of bee products have been discussed lately, being possible to treat several diseases simply and rapidly. There is nothing better for the body than the organic, biological products which can be found around us in nature, and in order to prove this hypothesis, we can go back to our origins: the miraculous features of bee products were registered even in the antiquity. Due to their components, bee cocktails are real “health sources”, taking into account the rich content in vitamins, minerals and other microelements, all these leading to its recommendation for multiple deseases. Being mixtures of beehive products in different formula, they help a lot our body which is so challenged by tiredness, stress, unhealthy food, pollution. The concept connected to nourishment has expanded from survival and satisfaction of hunger to the prevention of contrary effects with the use of these beneficial products to improve the quality of life and to prevent or reduce the risk of being taken ill. In order to adopt a healthy lifestyle, we are in front of a new way of dealing with the nourishing science: the use of the so-called “functional food”. Cosmetic products based on venom and other products of the beehive offer eternal youth due to the royal jelly (every day closer to a younger skin), the brilliance and delicacy of honey, the nourishing feature of pollen, the delicate texture of the natural bee wax, the cleanliness and protection given by propolis – the most powerful natural antimicrobial substance.

SECOND PART

Chapter 3 Personal research concerning the quality of bee products – raw material 3.1. Materials and methods

The analysis of the physical and chemical (a) and microbiological (b) composition of bee products which will be used in the bee therapy as functional food and in bee-cosmetics is extremely important because it is essential to know if products:

are genuine, have the adequate chemical composition to the declared type of bee product do not have pathogenic microbial load, and the saprophytic load is in the limits of

accessibility. (a) For the bee products which will become part of the suggested api-phyto-therapeutical

products, the main physical and chemical parameters were established: content of water, total proteins, total lipides (Table no. 6). The diastase index, electric conductivity and the content of HMF (hydroxi methyl furfural ) were also established because other physical and chemical characteristics are also important for honey (Table no. 7). The working techniques used comply with the monographs in FR X and the European Pharmacopoeia, the valid edition.

(b) The study of the antimicrobial activity of some bee products consists in establishing the antimicrobial features of bee honey, propolis, royal jelly, as well as establishing the efficiency of these products according to the antibacterial qualities in view of using them in medical attendance. Thus, the objectives of the microbiological study aim two stages:

1) screening the antimicrobial activity of bee products as compared to different bacterial and fungi stems

2) the quantitative determination of the minimum inhibitory concentration of the bee products tested as compared to different microbial stems.

The biological material. The study was achieved on 16 isolated microbial stems from patients in hospital. The identification of the stems was achieved with the automized system VITEK 2, as well as with the help of the microtest galleries API 20. The standard bacterial stems used in the study are:

a) Fungi stems: Candida albicans 103101, Candida famata 366, Candida albicans 601, b) Gram-positive bacterial stems: Enterococcus faecalis ATCC, Staphylococcus aureus

MRSA 349, SCN, Staphylococcus epidermidis, MRSA 1840, Staphylococcus aureus ATCC,

c) Gram-negative bacterial stems: Escherichia coli 634, Escherichia coli 8730, Klebsiella 136202, Pseudomonas aeruginosa ATCC, Acinetobacterbaumanii 7706, Pseudomonas aeruginosa 6633, Pseudomonas aeruginosa 719.

The tested bee products were:

Honey; Pollen; Royal jelly; Drone larvae; Propolis; Tincture of propolis; Soft extract of propolis

3.2 Results and discussions (a) The main physical and chemical parameters of bee raw materials were established: Table no. 6 The main physical and chemical parameters of bee raw materials used as raw material

No. Product Water content (%) Total proteins (% Total lipides (%)

1. Acacia honey 17.4 0.21 0.00 2. Manna honey 16.4 0.68 0.01 3. Pollen 2013 19.7 18.8 5.1 4. Pollen 2014 19.2 18.6 5.8 5. Maiden wax 15.7 16.6 4.4 6. Royal jelly 63.7 13.4 2.2 7. Apilarnil 61.4 7.6 3.2

The diastase index, electric conductivity and the content of HMF (hydroxi methyl furfural) were established for honey. Table no. 7 Physical and chemical characteristics specific to honey

No. Bee product Electric conductivity (mS/cm)

Diastase index (DN)

HMF (mg/kg)

1. Acacia honey 0.125 14.9 2.3 2. Manna honey 0.962 36.9 4.9

As far as one can notice in the analysis, acacia honey and manna honey are genuine products, having a high diastase index (the content of amylase proceeded from bees) and very low content of HMF. The electric conductivity of manna honey is high, over the minimum limit of the Romanian standard and Codex Alimentarius so that a type of honey could be declared manna honey (0.8mS/cm). For the main bee raw materials specific analysis were subcontracted by the laboratories of Physical-chemical and Microbiology Analysis of INCD IBA Bucharest (The Institute of Food Bioresources). The reports show the characteristics of raw materials (humidity, acidity, dried substance, total ash) according to valid STAS, but also pieces of information concerning the organoleptic features of bee raw materials: aspect, consistency, taste and smell, according to the monographs in the Romanian Pharmacopoeia, the valid edition (FR X). b.1. Establishing the antimicrobial activity of different bee compounds upon the analysed bacterial stems. A method adapted after the difuzimetrica one (Kirby-Bauer) was used. Conclusions: 1. The antimicrobial activity of bee products appreciated through the difuzimetrica method was reduced, areas of inhibition of the growth being slightly highlighted, except the tincture of propolis. 2. It has been noticed that in some cases, the antimicrobial activity of the tested products increased by the continuation of the incubation period (from 24 hours to 72 hours).

b.2. Establishing the CMI value (the minimum inhibitory concentration) using the method of microdilutions in a liquid environment. It allows to establish CMI expressed in U.I/mgr or gram product. Reading the results at spectrophotometer. The results were analysed by reading at spectrophotometer, too, this being a tool capable of measuring the quantity of photons accurately (the intensity of light) absorbed by putting them in a solution. Thus, the quantity of substance (concentration) may be established indirectly. For the stems of Candida albicans , all tested products have an antimicrobial activity, CMI has values between 1/10 and 1/80. The stem of C. famata proved to be sensitive only to the activity of pollen, the other compounds not having any effect. (Table no. 7.1 and 7.2) Table no. 7.1 The CMI and CMEB values of the bee products tested on stems of Candida sp

Code of the stem

Drone larvae

Royal jelly

Manna honey

CMI CMEB CMI CMEB CMI CMEB Candida albicans 103101 1/80 1/10 1/320 1/320 1/40 1/40 Candida famata 366 1/40 No effect No effect 1/10 No effect 1/80 Candida albicans 601 1/80 No effect 1/20 1/20 1/80 1/20 Table no. 7.2 The CMI and CMEB values of the bee products tested given the stems of Candida sp.

Code of the stem

Acacia honey Tincture of propolis

Raw pollen

CMI CMEB CMI CMEB CMI CMEB

Candida albicans 103101 1/40 1/10 1/320 1/10 1/80 1/40

Candida famata 366 No effect 1/20 No effect 1/10 1/160 1/10

Candida albicans 601 1/10 1/10 1/40 1/10 1/160 1/10

For the Gram positive stems analysed, drone larvae presented antimicrobial activity given all the stems, with an CMI value varying between 1/20 and 1/80μg/μl. The rest of the other tested products had an activity only to certain stems, with various concentrations, between 1/10 and 1/320 μg/μl (raw pollen given the SCN stems) (Table no. 8.1 and 8.2), the narrowest spectrum of activity being in the case of royal jelly.

Table 8.1. The CMI and CMEB values of the tested products given the Gram positive bacterial stems

Code of stem Drone larvae Royal jelly Manna honey

CMI CMEB CMI CMEB CMI CMEB

Enterococcus faecATCC

1/40 1/10 1/40 1/10 1/10 1/10

S. aureus ATCC 1/20 1/10 No effect 1/20 1/10 1/10 S. aureus MRSA 349 1/40 1/10 No effect 1/20 No effect No effect SCN 1/80 1/10 No effect 1/20 No effect No effect S. epidermidis 1/40 No effect No effect 1/20 No effect No effect S. aureus MRSA 1840 1/40 No effect 1/20 1/20 No effect No effect Table 8.2 The CMI and CMEB values of the tested products given the Gram positive bacterial stems

Code of stem Acacia honey Tincture of propolis Raw pollen

CMI CMEB CMI CMEB CMI CMEB Enterococcus faecalis ATCCNo effec No effect 1/20 1/10 No effect 1/10 S. aureus ATCC 1/40 1/10 1/80 1/20 1/160 1/10 S. aureus MRSA 349 No effec 1/10 1/40 1/10 No effect No effect SCN 1/10 1/10 No effect No effect 1/320 No effect S. epidermidis No effec No effect 1/160 No effect 1/320 No effect S. aureus MRSA 1840 1/80 1/10 1/80 1/10 1/160 1/10

For the Gram-negative analysed stems one can notice that drone larvae and the tincture of propolis have an antibacterial activity as compared to all analysed species, and the activity of the other compounds varies according to the analysed stem. The CMI values are between 1/10 and 1/320 μg/μl (Table no. 9.1 and 9.2)

Code of stem Drone larvae Royal jelly Manna honey

CMI CMEB CMI CMEB CMI CMEB

Pseudomonas aeruginosa ATC 1/80 1/10 1/40 1/10 1/10 1/10 Pseudomonas aeruginosa 719 1/80 1/10 No effect No effect 1/10 No effect Pseudomonas aeruginosa 6633 1/10 No effect 1/640 1/10 1/40 1/10 Acinetobacter baumanii 7706 1/60 No effect No effect 1/10 1/80 1/40 E. coli 8730 1/40 No effect No effect 1/10 No effect No effect E. coli 634 1/40 1/10 1/40 1/40 No effect 1/10 Klebsiella sp. 136202 1/80 No effect 1/10 No effect 1/10 No effect

Table no. 9.2 The CMI and CMEB values of the tested products given the Gram negative bacterial stems

Code of stem Acacia honey Tincture of propolis Raw pollen

CMI CMEB CMI CMEB CMI CMEB

Pseudomonas aeruginosa ATCC

1/20 1/20 1/10 No effect 1/320 1/10

Pseudomonas aeruginosa 71 No effect No effect 1/10 No effect No effect No effect Pseudomonas aeruginosa 66 1/40 1/20 1/10 No effect 1/160 1/10 Acinetobacter baumanii 7706No effect No effect 1/10 No effect 1/20 No effect E. coli 8730 No effect No effect 1/10 No effect 1/320 No effect E. coli 634 No effect 1/10 1/10 No effect 1/160 1/10 Klebsiella sp. 136202 1/10 No effect 1/160 1/10 No effect No effect

Chapter

4.1 Proc The analythe stand The contto the ana(1994) an Table no

No12345678

Figure no As one c1, there ithe Acachigh conestablishThe diffenot so big

r 4

essing raw m

ysis of the todard formu

tent of total falysis of beend adapted b

o. 10 Genera

o. Be1. Acacia h2. Manna h3. Pollen 204. Pollen 205. Maiden w6. Royal jel7. Drone la8. API PRO

o. 7 The con

an notice in s a very big ia honey. Th

ntent of mining the conteerence betweg, but the for

Bee

materials an

otal polyphela of bee Co

flavones wae products. Tby Meda and

al bioactive p

e product oney

honey 013 014 wax lly arvae ODUCT

ntent of total

Figure no. difference b

he much monerals make ent of polypheen the contrmer contain

cocktails (r

nd getting b

enols from thocktail was a

s establishedThus, the Dod col. (2005)

principles of

Total pol

polyphenols

between the ore complex

the manna henols provetent of total ns about thre

registered m

bee cocktails

he bee produachieved by

d by methodowd method.

f bee product

lyphenols (m17.71

108.00563.2612.4918.124.5243.51

1056.3

s of the type

content of tochemical cohoney be a

es that. flavones fo

ee times mor

mark OSIM)

s in the view

ucts used as y using the Fo

ds used in thd was used, m

ts and the be

mgGAE/100

es of honey a

otal polypheomposition, tan extremely

or the mannare flavones t

)

wpoint of IS

raw materiaolin-Ciocâlt

he specific litmodified by

ee cocktail p

Flavonoid

analysed

enols in the mthe dark coloy valuable b

a honey andthan the latte

SO 22000:20

als in order teu method.

terature , adArvouet and

product

des (mgQE/4.4 1.8

255.1 285.8 435.2 14.2 3.4

532.6

manna honeyour of ambebee product

d Acacia honer.

005

to get

apted d col.

/100

y and er, the t, and

ney is

Figure n

TcompounHPLC – the chromby the tim

Figure n

1

1

2

Abs

orba

nta

(m

AU

)

Figure n

no. 8 The con

The polyphends was achPDA (High

matogram reme of retenti

- 2 0 0 0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

1 8 0 0 0

Ab

sorb

an

ta (

mA

U)

no. 9 The HP

1 0

0

5 0 0 0 0

0 0 0 0 0

5 0 0 0 0

0 0 0 0 0

no. 10 The H

ntent of total

nolic profilhieved with t

Performancepresents oneion, the UV –

1 00 0 0

0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

PLC – PDA c

0 2 0

T im

HPLC – PDA

l flavones of

e of propolthe help of

ce Liquid Che or more po– Vis spectru

2 0

Aci

do

cum

aric

Aci

d fe

ruli

c

Aci

d p-

cum

aric

T im

Aci

d ca

feic

chromatogra

3 0

m p d e re te n t ie

A chromatogr

f the types of

lis as well the high pe

hromatograpolyphenolic um and the m

3 0

Kem

fero

l

Aci

d o-

cum

aric

Que

rcet

ina

p d e r e t e n t i e

am of the sam

4 0

(m in )

G a la n

ram of the sa

f honey anal

as the quaerformance cphy – Photo

compound, maximum o

4 0 5

Kem

fero

lC

AP

E

Gal

angi

naC

risi

na

Pino

cem

brin

a

( m in )

mple of alco

5 0

n g in a

ample of wa

lysed

antification ochromatograDiode Arrayand these cf adsorbtion

0 6 0

oholic propol

atery propoli

of the idenaphical techny). Each signan be recogn

n.

lis

is

ntified nique nal in nized

Identifying and quantifying sugars is an important step with a view to establishing the authenticity of samples both from a quantitative and a qualitative point of view. Table no. 11 The concentration of glucides (%) in the samples of bee products raw material and the apitherapeutical product achieved, established by the HPLC method

Sample Glucose Fruitose Saccharose Maltose Melezitoza (Sweet substance)

Total suga

Acacia honey 23.77 41.00 1.03 1.44 0.00 72.55 Manna honey 26.25 35.14 0.14 1.77 1.78 69.29 Pollen 2013 13.02 18.29 0.00 0.1 0.00 31.82 Pollen 2014 15.20 16.27 0.00 0.10 0.00 31.91 Maiden wax 9.23 17.264 0.00 0.23 0.00 28.66 Royal jelly 6.67 6.16 0.51 0.15 0.00 13.87 Drone larvae 8.68 8.76 0.14 0.32 0.19 19.00 API PRODUCT 19.49 27.08 0.01 0.95 0.47 50.21

5 10 15 20 25 30 35

0

10000

20000

30000

40000

zahar

oza

Abs

orba

nta

(mA

U)

Timp de retentie (min)

fructo

za

gluco

za

tura

noza

erlo

za

malt

oza

trehalo

za

Figure no. 12 The HPLC – IR chromatogram of the sample of royal jelly

5 1 0 1 5 2 0 2 5

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

Ab

sorb

an

ta (

mA

U)

T im p d e re te n t ie (m in )

gluco

za

zahar

oza

tura

noza

mal

toza

izom

alto

za

fruct

oza

Figure no. 13 The HPLC – IR chromatogram of the sample of drone larvae

2 4 6 8 10

-50000

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

Ab

sorb

ant

a (

mA

U)

T imp de retentie (min)

Apilarnil

Standard 10-HDA

Laptisor de matca

10-HDA

Figure no. 14 The overlapped chromatogram of the 10-HAD standard (40 mg/ml), of the royal jelly and drone larvae analysed In the composition analysis of royal jelly, one intended to establish the content of 10 DHA acid (10-HAD), which is a marker of authenticity of the royal jelly. Table no. 14 The antioxidant activity of bee products established by the DPPH and FRAP technique

No. Bee product DPPH (mmol TROLOX/g)

FRAP (mmol TROLOX/g)

1. Acacia honey 0.10 0.25 2. Manna honey 0.32 0.96 3. Pollen 2013 1.37 2.47 4. Pollen 2014 1.59 3.17 5. Maiden wax 8.64 11.42 6. Royal jelly 0.32 2.27 7. Drone larvae 0.68 16.41 8. API PRODUCT 6.34 10.03

As one can notice in the table above, both the total antioxidant potential and the activity of capturing the free radicals (DPPH) at bee products are high enough (as compared to other natural matrixes), and their combination in different ways leads to extremely valuable products such as their utilization in treating or even preventing various diseases. The explanation of the beneficial activities of bee products and the recommendation of their use in apitherapy, or medicine in general, is given by the detailed clearing up of the chemical composition of each of them. The variety of the composition is the decisive factor in the beneficial actions that these products have, the chemical analysis of their composition being their certificate of: guarantee, authenticity, quality. In order to comply with the valid GPM rules and the requirements for the safety of food, the ensurance of the traceability of the product from the beehive to the storehouse

or raw material, subsequently to its conditioning as a finite product as such or in different complex formula is required.

Fig no. 11 The traceability of Drone larvae from the beehive to the storehouse of raw material Materials and methods The quality of the drone larvae was checked by establishing: [1] the acidity – pH (5,9 – 6,9), [2] the humidity – water (65-75%), [3] the relative density (1,1 – 1,2), [4] the dried 10 g%), substance – residue (25 – 35%), [5] the ash (maximum 2%), [6] the reducing sugars (maximum [7] the total proteins (9-12 g %), [8] the total lipides (5-8 g %).

Pregătirea ramelor

Pregătirea familiilor de albine

Introducerea “ramelor clăditoare” (rame de control)

Verificarea şi corectarea spaţiului de ouare

Recoltarea ramelor în momentul optim

Extragerea APILARNILULUI

AMBALAREA

Depozitarea (congelarea) la apicultor

Pregătirea ramelor

Aspirare Stoarcere

Monitorizarea familiilor de albine

AP

ICU

LT

OR

UL

Pc 2

Pc 1

Pc 2

Pc 3

Pc 4

Pregătirea ramelor

Recepţia calitativă şi cantitativă

Transportul

Depozitarea la procesator

Procesare

Produs finit

Condiţionare

Depozitarea

PR

OC

ES

AT

OR

UL

Pc 5Pc 5Pc 5

Pc 6

Pc 7

Results and discussions In the process of getting the drone larvae, the authors appreciated that there are 7 checking points in all, out of which 4 checking points at the level of the operations performed by the beekeeper and other 3 checking points up to the moment where the drone larvae, as raw material, goes into processing. Table no.13 Results of the physical-chemical analysis of Drone larvae (samples M vs. stored product P)

M Proba pH Apa Densitate Rezidiu Cenuşa

% relativă % %

M Apilarnil 1 6.40 72.2 1.056 27.60 0.83

P Bihor 6.30 72.0 1.060 28.00 0.85

M Apilarnil 2 5.98 68.4 1.080 36.60 0.83

P Bihor 6.00 68.0 1.078 36.00 0.86

M Apilarnil 3 6.40 78.1 1.055 21.96 0.82

P Bihor 6.30 78.5 1.060 21.50 0.80

M Apilarnil 4 5.30 73.00 1.025 27.00 0.50

P Giurgiu 5.10 72.50 1.031 27.50 0.52

M Apilarnil 5 6.30 70.9 1.084 29.10 0.80

P Vaslui 6.28 65.8 1.081 34.20 0.90

M Apilarnil 6 6.20 59.1 1.140 40.90 0.71

Neamţ 6.10 57.2 1.170 42.80 0.72

Thus, from the first analysis, the supplier of the product Drone larvae 4 (Giurgiu) was eliminated because of the too high acidity of the product (pH 5.3), and the product Drone larvae 3 (Bihor) because of the too high humidity (78.1 %, compared to the limit of maximum 75% water indicated by the specific literature).Due to the high content of water, one could not establish quantitatively the total lipides of the samples: Drone larvae 2 (Bihor) and Drone larvae 6 (Neamţ). The suppliers of the two products were eliminated because of the high quantity of residue established (36.6 % with Drone larvae 2 and 40.9% with Drone larvae 6, compared to the limit of maximum 35% indicated by the specific literature). Finally, the products Drone larvae 1 (Bihor) and Drone larvae 5 (Vaslui) were selected, the results of the physical-chemical analysis of these products being at the limits allowed.The product Drone larvae 5 became conspicuous due to a relative density a bit higher (1.084 compared to 1.056) and to a higher content of reducing sugars and total lipides (2.5%, respectively 4.9% compared to 1.7%, respectively 1.96%), and the product Drone larvae 1, from the point of view of the content of total protein.

The antioxidant activity of the bee products Table no. 14 The antioxidant activity of the bee products established by the DPPH and FRAP technique

No. Bee product DPPH (mmol TROLOX/g)

FRAP (mmol TROLOX/g)

1. Acacia honey 0.10 0.25 2. Manna honey 0.32 0.96 3. Pollen 2013 1.37 2.47 4. Pollen 2014 1.59 3.17 5. Maiden wax 8.64 11.42 6. Royal jelly 0.32 2.27 7. Drone larvae 0.68 16.41 8. API PRODUCT 6.34 10.03

Aactivity oother natproducts Fof PhisicFood Biodried suconcernintaste and(FR X). Analysis Cocktail

Tadjuvantsproducts and royal Tproductsselective Adosage wpossible 4

B B B A

As one can nof capturingtural matrixfor their use

or the main cal, Chemicaoresources).

ubstance, totng the orga

d smell, accoSee the Anfor bee Coc

The modern s, in order toto the body.l jelly which

The mixture : honey, polly.

A system of dwere studiedallergies. bee cocktai

Bee Cocktail Bee Cocktail Bee Cocktail Apilife Apico

notice in the g the free radxes), and thee in treating

bee raw maal and MicroThe reports

tal ash) accanoleptic chaording to thennex no. 1 cktail, Anne

tendency iso increase th. Bee produch are compon

of bee prodllen, propoli

dosage was d, with the r

ls are listed of Immunityof Immunityof Immunity

ocktail

table above,dicals (DPPeir combinator even prev

aterials, specobiological s show the ccording to aracteristics

e monographReports of

ex no. 3 Phy

BEE s to use nathe therapeutcts are part onents of the ducts – BEEis, royal jell

established remark that

in the Api Ly for Womeny for Men y for Childre

, both the toPH) with beetion in diffeventing diffe

cific analysiAnalysis of haracteristicthe valid Sof bee raw

hs in the Romthe main b

ysical-chemic

COCKTAILtural productical arsenal of this categoBEE COCKE COCKTAly, drone lar

for the mixtno other ad

Life Ro portfn

en

otal potentiale products i

ferent ways erent disease

s were subcf INCD IBAcs of the rawSTAS, but

w materials: manian Phar

bee raw matcal and micr

LS cts both in and to offeory: honey, pKTAIL.

AIL is obtainrvae, maiden

ture of bee pdverse reacti

folio, namely

l antioxidants high enouleads to ex

es.

contracted byA Bucharest w material (h

also piecesaspect, con

rmacopoeia,terials, Annrobiological

prophylaxiser familiar, epollen, prop

ned by mixin wax in di

products, difions were no

y:

t (FRAP) anugh (comparxtremely valu

y the laborat(The Institu

humidity, acs of informnsistency, co the valid ed

nex no. 2 Hl analysis fo

s and as treeasily assimiolis, drone l

ing differenfferent quan

fferent systemoticed excep

nd the red to uable

tories ute of cidity, mation olour, dition HPLC or bee

eating ilable arvae

nt bee ntities

ms of pt the

Chapter

Gonly a soas the mabe eaten Btherapeutthe categand the ias well atraditiona ARONI process melanocaBee honecharacter

CĂTIN underbruBee honcharacter

r 5

Generally, thource of foodain bee prodas having sp

Bee honey istical charact

gory of functntegrative ms in our counal medicine

APILIFE –of precocio

arpa (figure ey combinedristics of “fohelps to invhas a benefblood pressureduces the it is recommit is beneficthe level of it contributeit is an exce

APILIFE ush (figure noney combineristics of “fo

contributesit is a geprotector; it helps chiit helps theeffort, tired

Functionae products o

d, but also spduct was recpecial characs a type of teristics. Thetional food?”

medicine, basntry, honey for a long tim

– a powerfuous ageing.no. 19) d with fruit od with specigorate the i

ficial effect oure; oxidable str

mended to prcial for the dglucose in th

es to the detoellent genera

– it fortifieo. 20) ed with theod used to k

s to the compeneral, vitam

ldren develoe body in stdness.

al food withof the beehivpecial remedommended acteristics, favf food whiche following ” The answesed on evideand the otheme”.

ul antioxidan. It contain

of Aronia mcific use for mmunity syon blood cir

ress and makregnant womdiabetes of tyhe blood; oxification ol tonic

es the body

e underbruskeep healthy”pleting of numinizing, a

op normally tressful situ

h the producve represente

dies against das “a primarvourable to hh best combquestion is er would be:ence.” A super products o

nt, it invigorns: bee hon

melanocarpakeeping heastem;

rculation, he

kes the procemen due to its

ype 2 due to

of the body;

y. It contain

sh fruit giv” which:

utritive deficantianaemic

and harmonations, nerv

cts of the beed for peopldifferent disery food subshealth and th

mbines the nasked: “Cou: “Definitelypplementaryof the beehiv

rates the imney with d

a give the pralthy” which

eart function

ess of precocs rich conteno the presen

ns: bee hon

ves the pro

iencies; tonic, a c

niously; vous challen

eehive le, even in aeases. In antstance” and ihe prolongatnourishing quld bee honey, based on ly reason: “ Inve have enter

mmunity systdehydrated

roduct AROh:

ning and the

cious ageingnt of folic acnce of inulin

ney with deh

oduct CĂTIN

coronary, an

nge, intellect

ancient timesiquity, bee hit was allowtion of life. qualities withey be includlaboratory ren other counred the arsen

tem, preventfruit of A

ONI APILIFE

normalizati

g slow down;cid; , it may dec

hydrated fru

N APILIFE

ntiatheroscle

tual and phy

s, not honey

wed to

h the ded in esults ntries, nal of

ts the Aronia

E the

on of

;

crease

uit of

E the

erosis

ysical

CAIS APand the dapricots. Bee honeused to k

n su pr pr h

The prepsome stuvitamins

COACĂZstimulategooseberBee hone“food use

hb

gp

s i

ti

si

PILIFE – it ediminution o(figure no. 2

ey combinedkeep healthy”ormalizes thupports cardrevents the arevents osteoas a tonic ef

paration of thudies which

and, in wint

Z APILIFE es the corticrry bush (figuey combineded to keep hhelps duringbody; general toniphysical andstimulates hait is a naturathe corticoainflammatorysupports the improves the

ensures cardof visual acu21)

d with aprico” which:

he intestinal tdiovascular happearance ooporosis;

ffect, removehe product Cproved that

ter, they get

– helps thcoadrenal fuure no. 22). d with apricealthy” whic

g the ………

c effect in d intellectual aematopoies

al product widrenal funcy and antiallnormal func

e visual acui

diovascular puity, it is a g

ots gives the

transit; health; of cataract;

es physical aCAIS APILIt: “althoughthe value of

he body recunction. It c

ots gives thch: ……. cures

conditions efforts;

sis; ith an effect ction and relergical effecctioning of tty.

protection, fogood laxative

e product CA

and psychic aIFE as funct

h dried, apricf medicine”.

cover after contains: bee

e product C

by eliminat

of asthenia,

similar to theleasing somct; the liver, the

ortifies the be. It contain

AIS APILIFE

asthenia. tional food wcots keep th

intense phye honey wit

COACĂZ AP

ting the toxin

, tiredness,

he cortizon-lme substanc

e pancreas, t

bony systemns: bee honey

E the charac

was based oheir content

ysical and inth dehydrate

PILIFE the

ns and the u

for recover

like hormonces with a

the spleen an

m, prevents agy and dehyd

cteristics of “

on the remarof minerals

ntellectual eed fruit of b

characteristi

uric acid from

ring after in

nes by stimulremarkable

nd the kidne

geing drated

“food

rks of s and

effort, black

ics of

m the

ntense

lating anti-

eys; it

GOJI APimmunity Bee honused to k

imre

It It It It

m It

ca It It It

m G

PRUNAPintestinal24) Bee hon“food use

Istr

C E Im It

o

PILIFE – givy, increases

ney combinedkeep healthy”mproves the etina. t is an import increases tht regulates tht has a bene

minerals and t protects thalcium; t is hepatoprt helps to balt has a favou

memory; Gives energy

PILIFE – nol transit, a n

ney combineed to keep hs a powerfulransit and no

Contributes toEncourages dmproves the t has nourishf tiredness.

ves energy alongevity. Itd with apric” which: disorders of

rtant source ohe capacity ohe level of chficial effect vitamins;

he health of

otector and ilance the horurable influe

and vitality

ourishing chnatural invigo

ed with apriealthy” whicl laxative anormalizes theo the regulat

detox and cleliver conges

hing charact

and vitality, t contains: beots gives the

f eyesight, p

of antioxidanof natural deholesterol; on conditio

f bones and

it has anti-agrmones, it imence upon c

, it ensures g

haracteristicsorator. It co

icots gives ch: nd an excellee intestinal fltion of the aceaning the costion; eristics, it is

protects the ee honey ane product GO

prevents the

nts which sloefense of the

ons of anaem

d ligaments,

geing characmproves the conditions of

good mood.

s, an excellentains: bee h

the product

ent digestiveflora; ctivity of panolon;

s naturally fo

body againsd dehydratedOJI APILIFE

diminution

ow down thebody;

mia due to it

, especially

cteristics; sexual functf anxiety, st

ent digestivehoney and d

t PRUN AP

e stimulus w

ncreas and b

fortifying and

st oxidating d goji fruit (E the charac

of visual ac

e process of

ts high conte

due to its

tion; tress, tiredne

e stimulus, adehydrated p

PILIFE the c

which regula

body weight;

d drives awa

stress, stimu(figure no. 23cteristics of “

cuity, protect

f ageing;

ent of iron,

high conte

ess and impr

an adjuster oplums (figur

characteristi

ates the inte

;

ay the condi

ulates 3) “food

ts the

other

ent of

roves

of the re no.

ics of

stinal

itions

ZMEUR silhouetteBee hone“food use

Ssy

Itpr

It It It It

MINT Aconditionno. 26). Honey ccharacter

co P It It

Results aTable nohoney wi

The limit oadmissibiliHoney with

Honey with

Honey withUnderbrus

APILIFE –e. It containsey combineded to keep hupports the ymptomps ot is a way oressure, the t reduces thet has a positit improves sot has nourish

APILIFE – ns of nausea

combined wristics of “foontrols abdorevents the ft soothes cont controls dia

and discussio. 15 The coith powder o

Total no. aerobian microorga(TAMC) U

of ity

104

h Aron 7,5 x 101

h apric 5,0 x 101

h sh

< 10

– improves ds: bee honeyd with apricealthy” whicnormal func

of constipatiof maintaininarterosclero

e appetite, beive influenceome feminin

hing, tonifyin

a powerful a and vomiti

with the chood used to k

ominal distenfermentativenditions of varrhea and n

ions ontent in actof vegetal ma

of

anUF

Total combinof fibreous leaand fungi(TYUFC/g 102

2,0 x 101 (Asp. flavus) 0,5 x 101

< 10

digestion, coy and dehydrcots gives thch: ctioning of thon; ng the cardiosis. eing useful ine upon the bone endocrineng characteri

source of ng. It contai

opped leavekeep healthy”nsion, helps e intestinal pvomiting andormalizes th

tive principlaterial (fruitsea

YMEnterobactceae UFC/g

Spe

Staphcoccu

102 abse

absent abse

absent abse

absent abse

orrects the frated raspberhe product Z

he gastro-int

ovascular sy

n preventingody sensitive

e disorders, iistics and vit

vitality, it ins: honey a

s of mint g” which: to eliminate rocess;

d nausea; he intestinal t

les and micrs and medicicific microorgani

hylo us aur

Escherichia

ent absent

ent absent

ent absent

ent absent

feminine enrry (figure nZMEUR AP

testinal tract

ystem, it pre

g fatness; e to repeatedirregular pertamins.

stimulates dand fresh ch

give the pr

the winds in

transit.

robial containal plants)isms Co

pec [%

a Salmonelaspp.

absent

absent

3,3

absent

-

absent

3,3

ndocrine disoo. 25)

PILIFE the c

and improv

events the ar

d colds, it deiod;

digestion, chopped leave

roduct MEN

n the stomac

amination fo

ontent in ctines

%]

ContenExpreschlorid[mg/10

35 8,1

-

32 -

orders, keep

characteristi

ves the unple

rterial high b

ecreases feve

controls diares of mint (f

NT APILIFE

ch and intest

or the sampl

nt ssed in de of , 00g]

Contecaroteexprescaroten

-

- 3,94

ps the

ics of

easant

blood

er;

rrhea, figure

E the

tines;

les of

ent enes, ssed in n, [mg/1

Honey with Gooseberry

< 10

< 10 absent

absent

absent

absent

3,92

17,6

-

Honey with Goji

7,0 x 101

0,5 x 101 (Asp.niger)

absent absent

absent

absent

2,91

-

0,65

Honey with plum < 10

3,0 x 101 absent

absent

absent

absent

6,66

They identify; theycan’t be measured

0,008

Honey with Raspberry

1,1 x 101

0,5 x 101 absent

absent

absent

absent

6,91

2,2

-

Honey with Mint 2,5 x 101

1,0 x 101

absent absent

absent

absent

1,35

5,1

-

All samples are adequate. They joined the limit of admissibility stipulated by the valid European Pharmacopoeia. The samples of honey with Goji and Aronia join the limit of admissibility, but they are contaminated with Aspergillus niger. The samples are not contaminated with specific pathogenic microorganisms. Chapter 6

Preparations with bee venom Bee venom, raw material submitted to analysis The characterization of the main proteinic fractions in the Romanian bee venom was achieved with the help of the high quality liquid chromatography and mass spectrometry. This approach was chosen in order to get valuable pieces of information (qualitative and quantitative) concerning the possible differences existing in the composition of the samples with bee venom with a different origin and collecting year. The main aspects followed in the proteinic characterization of the venom were:

stamp confirmation of the quality authenticity origin

Materials and methods The origin of the venom samples was from Romania and Ukrainia, and they were collected in 2013. The samples were kept in the dark up to the moment of analysis, at a temperature of 5°C, and the data of identification of the samples were for: V1 –Gorj, Romania / V2 –Bihor, Romania / V3 –Sibiu, Romania / V4 –Neamţ, Romania / V5 – Ukrainia / V7 – Ukrainia. The analysis of the venom samples with high performance chromatography of liquids, HPLC was done in the laboratories of The National Institute of Research-Development for Cryogenic and Isotropic Technologies – ICSI Rm. Vâlcea. The quantification of the components of venom (melitin, apamin, phospholipase A2) was intended by using the method of the external standard using standards of reference of analytic purity, Sigma-Aldrich. The samples of venom and the standards were prepared in acidulated water with TFA. The chromatographic conditions were fulfilled by using:

HPLC Thermo Finnigan Surveyor equiped with a detector row of diodes (PDA) Separation – the Aquasil C18 column (250 × 4.6 mm, 5 μm) PDA detector set at the wave length of 220 nm The elution in gradient of two mobile stages (water and acetonitryls acidulated with TFA

– trifluoroactic acid)

Figure n Testablish Bruker Msupport pfollowedthose in tResults a Treal samp Tvenom sa

Figure n

no. 27 High p

The analysis the profiles

Microflex LTprogramme od by the statthe data baseand discussi

The HPLC mples, and the

Table no. 16amples

no. 28 The co

performance

of venom / proteic staT spectromeof control Cotistical analye “Apitoxin ions

method of ese quantitative

6 The quantit

Compus

Apamina

Fosfolipaza

Melitina

omposition o

e chromatogr

samples by amps by maseter from Brompass 1.3 fysis of theseTaxonomy”

tablishing the and percen

tative and pe

s V1

a 4.51

A 21.7

a 60.5

of the venom

raph of liqui

mass spectss spectromeruker Daltonfor FLEX v3e. The purc(Ionete et a

he proteic contage results

ercentage co

Pr

V2 V3

4.74 4.1

23.4 21.8

62.5 60.4

m samples an

ids, HPLC S

trometry MAetry. The wonics Inc. (B3.3 . The acq

chased spectal, 2013).

ontent of veare rendered

omposition o

roba (%)

3 V4

19 4.56

8 23.1 2

4 65.4 6

nd the HPLC

Surveyor Plu

ALDI-TOF orking equipBillerica, MAquisition of tra were int

enom sampled in the Tabl

of the active

V5 V7

4.03 4.14

20.1 20.6

62.2 62.9

C chromatog

us

was intendpment was a A, USA) , wmass spectraercompared

es was applile no. 14.

e principles i

gram

ed to mass

with a a was with

ied to

in the

The samseparatiocomparedpurificati

Figure n

Figure ndone for

mples presenton of the cod to the inteion – as it is

no. 29 The ch

no. 30 The seeach analyti

t a similar components egral venomcollected by

hromatogram

election of thic sample of

content of aof apitoxin

m (no mattery beekeepers

ms for a) apa

he most reprf standard an

apamin, phos(melitin, a

r if it is puris) and to hon

amin, b) pho

resentative sd apitoxin

spholipase Aapamin, phoified or in iney.

ospholipase A

spectra in th

A2 and meospholipase its natural co

A2 c) melitin

e multitude

elitin; an obvA2 ) is noondition, wi

n

of measurem

vious oticed ithout

ments

An obviofresh sam Tof 0.6 mg Cgroupingvenom sa Practica W 6.1 Revit

IngredienMentha pmethylisoPresentathe proceled to thereducing circulatioarticular acids, vitbee venantiinflammakes atransepidantiinflamsupplenefactors frAction: Dof hot pehas an amuscularand the hWay of w30 ml, 5distinguiat the sam

ous separatiomples of bee The analysis g/g for apam

Concerning thg in the samamples – we

al results We go on wit

talizing gel

nts (INCI): piperita oil,othiazolinonation: A speess of biologe stimulationfine lines a

on, to decrepains. The

tamins, phyom, the exmmatory, soa very finedermical wammatory andss, firmnessrom the enviDue to its ricepper and mntiinflammar pains or duhealthy aspecwrapping u0 ml and labshing marksme time the i

on of the purvenom. of venom sa

min, 11.4 mg/he dispersio

me area of te can definite

th the presen

with bee ve

Aqua, Glyce Pinus sylvee, Bee venom

ecial productgical and pren of the pro

and wrinklesease musculproduct is b

ytohormonesxtracts of hoothing, reve film/layer ater. Revitd soothing es and protecironment. ch content o

mint and pineatory effect, ue to other cact of the skinp. The produbelled with

s, validity terintegrity of t

rified venom

amples by m/g for phosp

on of the venthe diagramely say that i

ntation of AP

enom, hot pe

erin, Capsicestris oil, Bm t, with bee vemature ageoduction of c, to increasear, sciatic pbased on na, enzymes, hot pepper,italizing and

on the sualizing effe

effects for thction to the

of natural che essential o

stimulates bauses, neuran. uct is wrappthe name o

rm, etc. Thethe product t

m (lyophiliza

mass spectromholipase andnom sample

m, without hait is natural (

PILIFE cosm

epper, mint

cum frutesceBenzyl alcoh

venom, createing of the bcollagen ande the permeapains and toatural bioactproteins, am, mint and d emollient urface of thects, of rehe cutaneous

skin agains

emical compil, the produblood circullgia, bruises

ped in jars mf the produc wraps havethroughout t

ated powder)

metry MALDd 9.3 mg/g fos from beekaving a diff(fresh) bee v

metic produc

t and pine

ens extract, ol and meth

ted to care abody. The red elastin, it ability of theo reduce netive compou

mino-acids, md pine esse

characteristhe skin, thejuvenation s tissues andst the aggre

pounds fromuct helps thelation, it is s, strains, fra

made of plastct, ingrediene an elegant the whole pe

) is also noti

D led to the ofor melitin. keepers , onefferent separvenom.

cts

Carbomer, hylchloroiso

and upkeep esults of the helps to rege capillary veuromialgia unds: flavonmicroelemen

ential oils wtics, in a bahus prevent

of the sd the inflameessive action

m the bee vene muscles anbeneficial f

actures, it ke

tic with a lidnts, action, pand modern

eriod of mini

iced compar

obtained qua

e can notice ration among

Triethanolamothiazolinone

the skin liablocal applic

generate the vessels and b

, rheumaticnes, polycarbnts, etc fromwith nourisase of gel wting the losskin, pain-ked joints, it n of the har

nom, the extnd joints relfor the rheumeps the resil

d, of a capacproducer, grn design, ensimum durab

red to

antity

their g the

mine, e and

ble to cation

skin, blood c and boxyl m the shing, which ss of killer, gives rmful

tracts lax, it matic lience

ity of aphic

suring ility.

Usage: Sabsorbs rthe pain. with mycto bee stismall surthe produWay of place, aw This proOSIM wpublished 6.2. Rege

Ingredienalcohol aPresentaformationbee subsenzymesemollientthus prevdeep layethe aggreAction. circulationormal lithe firmnthe supplWay of wcapacity produceringredienbefore...”time the Usage: Tthe eveniWay of pwrap, in

Soft, local aprapidly and c

Notices: Dcosis and infings, pollen rface of the suct may be upreserving:

way from ligh

duct is the fwith the appd in BOPI 12

enerating n

nţi (INCI): and methylchation: A specn. That is wstances, bee , proteins, t features, in

venting the lers of the epessive actionIt activates

on at the peimits, prevenness and theleness and fiwrapping upof 15 ml, 30

r, address, tents, an indiv”, conventiointegrity of t

The product ing, by a sofpreserving:clean, dried

pplications oncompletely, o not let it a

flammationsand pregnan

skin (1-2 cmused accordin These prodht and heat.

final, improlication no. 2/2013 , see

ourishing fa

Aqua, Glychloroisothiazcially createhy the skin venom andfree amino

n a base of gloss of transepidermis, whn of the harm the cellula

eripherical lents prematurtonus of th

irmness of thp. The produ0 ml, 50 ml, elephone numvidual bar cnal signs. Tthe product tis for extern

ft massage w The producspaces, awa

nce up to thrit offers a feat hand and . Bee venomnt women. B

m²), one has tng to the insducts are to

ved and not2012 0028Annex no. 7

ace gel with

cerin, Carbozolinone and

ed product tomust be cared royal jellyo-acids, micgel which maepidermical

hich gives sumful factors far regeneratevel, keeps re ageing ane skin. It ha

he skin. It douct is wrappinscribed ac

mber, fax, node, the num

The wraps hathroughout t

nal use, it hawith the top fct is stored aay from heat,

ree times a deeling of fresight of you

m must be avBefore the fito wait for 4tructions. be used onl

tified in the5 on the 257.

h bee venom

omer, Royald methylisotho care sensitied correctly y, rich in vcroelements,akes a very fwater, ensu

uppleness, firfrom the envtion, feeds the lipo-hy

nd delays theas a fortifyinoes not have ed in jars maccordingly (tominal contmber of theave a speciathe whole peas to be applifingers. at temperatur, cold and hu

day on the afshness, contung children

voided by astirst use, the 48 hours and

ly externally

e European U5th April 20

m and royal j

l jelly, Tiethhiazolinone,ive skin, liab and efficien

vitamins, ph, etc. with fine film/lay

uring the tranrmness and p

vironment. and fortifiedric balance

e natural ageng, velvety,

unpleasant rade of plastithe name oftent (ml), thee lot, the noal, modern deriod of validied in a thin

res between umidity.

ffected area,tributing to tn. It is unadvthmatic peopproduct wil

d if there is n

y and they a

Union of th012 the dep

jelly

hanolamine, Bee venom

ble to the prontly. The pro

hytohormonenourishing

yer on the sunsport of theprotection to

es the skin,e and the pHeing of the skrelaxing effreactions upic with an opf the producte sign of the

otice “To bedesign, ensudity.

n layer, in th

10 and 25 °

in a thin laythe attenuativisable to pple, those allll be appliedno adverse e

are kept in a

e gel patentpositing date

Parfum, Bm

ocess of wrioduct is basees, a compleg, hydratingurface of the e nutritives io the skin ag

activates bH of the skkin, reestabl

fect and incrpon the skin.perculum lidt, the name oe recycling we used prefeuring at the

e morning a

°C, in its ori

yer. It ion of eople lergic

d on a effect,

a cold

ted at e and

Benzyl

inkles ed on ex of

g and skin,

in the gainst

blood kin in lishes reases

d, of a of the wrap, erably same

and in

iginal

6.3 Solut

IngredienBenzyl alPresentareduce ncompounmicroelemnourishintransportAction. Dpepper, mantiinflamor due toaspect ofWay of wwith a spdistinguiproduct tUsage: Sabsorbs rpain. Way of pclean, co Avenom, a Cand allerwith beevenom anonallerg

tion of bee v

nţi (INCI): Alcohol and mation: A speneuromialgiands: flavonesments, etc fng, antiinflat of phytocomDue to its rimint and pimmatory effo other causf the skin. wrapping upprayer and itshing marksthroughout thSoft, local aprapidly and c

preserving: ol places, aw

A survey on at The authoClinical survergic reactione venom, hoand royal jelgic volunteer

venom – spr

Aqua, Capsimethylchloroecial producta , rheumatis, polycarbofrom the beeammatory, sompounds in tich content oine essentialfect, stimulatses, neuralgi

p: The prodt is labelled s, validity terhe whole perpplications completely,

The productway from lig

the irritabiliorised Dermoeys of testinns were donot pepper, mlly” and “Srs, men and

ray

icum frutescoisothiazolint, with bee vic and articxyl acids, vi

e venom, theoothing, revthe deep layof natural chl oils, the ptes blood ciria, bruises, s

uct is wrappwith the na

rm, etc. The riod of mini, once to thit offers a f

t is stored atght and heat.

ity of the sko Test Păung the cutanee for all the

mint and piSolution of b

women, the

cens extract, one and met

venom, creatcular pains. itamins, phye extracts ofvitalizing anyers of the ephemical com

product helprculation, it istrains, fract

ped in contaiame of the p

wraps are emum durabi

hree times a feeling of fre

t temperature

kin was donena SRL laboreous tolerance three prodine”, “Regenbee venom –final report

Mentha pipthylisothiazoted to decreaThe produc

ytohormonesf hot peppernd emollientpidermis. mpounds frops the musclis beneficialtures, it kee

iners made oproduct, ingreasy to use aility. day, on the

eshness, con

es between 1

e for all the ratory, Annece from the pducts with benerating no– spray” onis found in t

perita oil, Pinolinone, Beease musculact is based s, enzymes, pr, mint and pt features, in

m the bee vles and joinl for the rheueps the resil

of plastic witredients, actiand they assu

e affected arntributing to

10 – 25 °C, i

three cosmeex no. 8. point of viewee venom: ourishing fan a lot of 20the Annex n

nus sylvestrie venom ar, sciatic pa

on natural proteins, ampine essentian water, ass

venom, extrants to relax, umatic muscience and th

th a capacityion, produceure the integ

rea, in a thinthe attenuat

in its origina

etic products

w of the irrit“Revitalizingace gel with0 people, heano. 9.

is oil,

ains and to bioactive

mino-acids, al oil with suring the

acts of hot it has an

cular pains he healthy

y of 50 ml, er, graphic grity of the

n layer. It tion of the

al wrap, in

s with bee

tative g gel

h bee althy,

Chapter 7 Conclusions and perspectives

7.1 General conclusions The theoretical purpose of the thesis was to evaluate the qualitative (nourishing and therapeutical) value of the products of the beehive and, at the end, their practical utilization in the shape of preparations:

Bee cocktails Functional food Cosmetic products

From the point of view of the food safety, it was proved that bee products, in order to be used in apitherapy, must strictly respect the collecting conditions, conditioning and storage. By their chemical nature (the main components being glucides, proteins, vitamins, enzymes) they are a favourable environment for the development of microorganisms, so they present the risk of microbial loading. In order to respect the valid GMP (Good manufacturing Practice) rules and the requirements for the safety and security of food, one must ensure the traceability of the bee product from the beehive to the warehouse of raw material, subsequently to its conditioning as a finite product (as such or in different complex formula), by implementing an Integrated System of Management (of the food Quality, Environment, Safety and Security) on bee farms, in the factories of processing and producing bee products, which allows the notification and correction of risky factors by analysing the Critical points of control (Cpc). The survey on “Studies and research concerning bee products. Utilization in the shape of bee cocktails, functinal food and cosmetic products based on bee venom” had in view the documentation, experimentation and evaluation of some technological alternatives to get some complex food and cosmetic products based on the products of the beehive, in view of the optimization of the:

chemical composition (by association) way of preserving effect on health.

The importance of the chemical components in the bee products was taken into consideration to keep and improve health, as well as the curative features of fruits and medicinal plants (reinvestigated and used with the help of modern technology), having in view to get some “api-phyto” like products therapeutically efficient at the level of the human body. We think that the aims of the survey were reached, having gone through all the stages established in the protocol of work (documentation, research, manufacture and test). Thus,

1) the most important products of the beehive from the point of view of the chemical qualitative composition were studied in the first stage, subsequently doing the quantitative analysis of the components with a beneficial effect on the body.

2) the aim of the second stage was to get some api-phytotherapeutical products such as “bee cocktail”; various technological ways of preparation were established and best ways of preserving and dosing were identified.

3) the third stage had in view to study the possibility of getting some functional food. Thus the range of functional food “APILIFE” was obtained, based on bee products which, in association with fruits and medicinal plants, help to keep and improve health.

4) the fourth stage, the last one, ended with the formulation and test of a range of cosmetic products based on bee venom, simple or associated with medicinal plants.

7.2 Recommendations As a beekeeper, the author of this thesis has studied the methods of collecting bee venom for 10 years (2004 – 2014), noticing that the stimulation of the aggressiveness of bees establishes the growth of the collected quantity of venom. Based on her acquired experience, the PhD student Engineer Cornelia Abălaru (Doşteţean) is entitled to remark the following:

the aggressiveness of bees can be stimulated without harming the physiology of the bee and the activity of the beehive

the growth of the collected quantity of venom is directly proportional to: the power of the bee family the existence of sufficient resources of food in nature the use of the collecting technology of bee venom

recommending the improvement of the monitoring apparatus and the devices for collecting the bee venom in view of their optimization. 7.3 Personal contributions and future tendencies of research development The PhD student:

made the analysis laboratories of ICSI Romania, USAMV Cluj, ICD Beekeeping, INCD Food bioresources Bucharest, SC Hofigal Export Import SA Bucharest, etc. available, various samples of products of the beehive (from different places in the country and abroad, collected in different years) thus the main components being able to be analysed.

being interested in the improvement of the technologies of getting bee products, especially in the collecting of bee venom and the permanent monitoring of its chemical parameters (melitin, apamin and phospholipase A2), she initiated the creation of a database and the implementation of a work method (validated by ICSI Romania) to analyse the authenticity and quality of venom (an essential stage in the standardization process of bee venom, necessary to all beekeepers and apitherapists because there is no Standard of product for the bee venom).

by collecting the bee venom since 2004, she succeeded throughout the 10 years of experimentations and testing in improving the apparatus and method of harvesting, reaching the performance of 2014 of harvesting 2-3 g/day/20 families (compared to the first year when only 6 g/the whole bee season/ 20 families was harvested).

as a legal representative of the economic agent SC Apilife Ro SRL Sibiu she got the registration at OSIM as trademark the product/the name “Bee Cocktail” in view of ensuring a better control of the market outlets of the mixtures of bee products with therapeutical use.

by documentation and testing, she succeeded in perfecting the way of preparing bee mixtures like cocktail and functional food. By perfecting the way of conditioning raw materials (bee/fruits/plants) and the work method she succeeded in getting a stable, homogeneous finite product which no longer separates in phases.

she created 3 cosmetic products based on bee venom which are now being notified by the European procedure harmonized on the portal CPNP under the commercial names of:

Revitalizing gel with bee venom, hot pepper, mint and pine Regenerating nourishing face gel with bee venom and royal jelly Solution of bee venom - spray

and the dermatological testing done at an accredited laboratory classified the products in the category of Creams, emulsions, lotions, gels and oils for skin – face, hands, feet – with their classification in the category UNIRRITATING. The purposeful practice of the research activity in the project was to get 3 categories of api-phytotherapeutical products :

bee cocktails functional food

cosmetic products The results obtained are thought to be of interest, both on the national and the international market, in the field of

traditional food products food supplies cosmetic products

based on products of the beehive. We consider that due to the positive results obtained through this reference study of fundamental research with a practical purpose, from a scientific point of view, there are encouraging premises for other patent application to be registered at OSIM in the near future concerning:

products procedures methods devices

in the field of beekeeping, food industry and api-phyto-therapy with a value at the national level. For the reasons mentioned, after having presented her PhD thesis in a public meeting and its confirmation as being in accordance with the current legal conditions related to intellectual property, the PhD student will revise the study in the sense of mentioning in a wider and more detailed way several original aspects of the invention type. The author also decided to continue and study thoroughly the research concerning the ways of getting bee products and their utilization in perfected complex mixtures as a nourishing and therapeutical contribution. The results obtained will be communicated during national and international scientific manifestations, they will be disseminated with the help of printed publications and on computer in order to ensure a continuity of the research direction, by completing and bringing up-to-date with many other innovating, original and prior ideas, so that the general and endorsed public should have the possibility to apply directly and in a more pragmatic way all these scientific, theoretical and practical innovations which contribute to the progress of knowledge and social, bioeconomic and lasting development. Bibliografie selectivă 1. BĂRNUŢIU TOMOŞ L., 2013, Evaluarea proprietăţilor biologice ale markerilor de calitate

din lăptişorul de matcă şi apilarnil, Teză de doctorat, USAMV Cluj-Napoca 2. GIANFRANCO DILetti, 2006, Chloramphenicol in royal jelly: analytical aspects and

occurrence in Italian imports., Apidologie 37,6., 673-678 3. ILIESIU V.V., (1991), Apilarnil o nouă sursă apicolă de substanţe biologic-active, în folosul

sănătăţii omului, Editura Apimondia 1991 4. IONETE R. E., DINCĂ O. R., TAMAIAN R., GEANĂ E. I., 2013, Exploring Apis

mellifera Venom Compounds Using Highly Efficient Methods. Progress of Cryogenics and Isotopes Separation 16(2):89-100

5. KIM JOONYEONG ŞI JONGSEOK LEE, 2010, Quantitative analysis of trans–10-hidroxy-2-decenoic acid in Royal Jelly products in USA by high performance liquid cromatography, Jornal of Apicultural Science 54(1), 77-85.

6. KOKOT Z.J., MATYSIAK J., (2009), Simultaneous determination of major constituents of honeybee venom by LC-DAD. Chromatographia 69:1401–1405.

7. KOKOT Z.J., MATYSIAK J., KŁOS J., KĘDZIA B., HOŁDERNA-KĘDZIA E., (2009), Application of principal component analysis for evaluation of chemical and antimicrobial properties of honey bee (Apis mellifera) Venom. Journal of Apicultural Research 48(3) 168-175. DOI: 10.3896/IBRA.1.48.3.04.

8. KOKOT Z.J., MATYSIAK J., URBANIAK B., (2011), New CZE-DAD method for honeybee venom analysis and standardization of the product, Anal. Bioanal.Chem. 399:2487-2494. DOI: 0.1007/s00216-010-4627-2.

9. LAZĂR ŞT., VORNICU C.O., (2007), Apicultura, Editura Alfa, 656 pag.94 10. SABATINI, A.G., MARCAZZAN, L.G., CABONI, M.F., BOGDANOV, S., ALMEIDA-

MURADIAN, L.B., 2009, Quality and Standardisation of Royal Jelly. Journal of ApiProduct and ApiMedical Science 1(1):1-6.

11. STÂNGACIU S, 1999, Apiterapy course notes. Bucuresti, 12. YATES J.R., RUSE C.I, NAKORCHEVSKY A., (2009), Proteomics by mass spectrometry:

Approaches, advances, and applications. Annual Rev Biomed Eng 11:49-79. 13. YUCEL, B., ACIKGOZ, Z., BAYRAKTAR, H., SEREMET, C., 2012, The effects of Apilarnil

(Drone bee larvae) administration on growth performance and secondary sex characteristics of male broilers, Journal of Animal and Veterinary Advances, 10(17), 2263-2266.

14. ZHOU J., ZHAO J., ZHANG S., SHEN J., QI Y., XUE X., LI Y., WU L., ZHANG J., CHEN F., CHEN L., (2010), Quantification of melittin and apamin in bee venom lyophilized powder from Apis mellifera by liquid chromatography-diode array detector-tandem mass spectrometry, Anal Biochem. 404(2):171-178, doi: 10.1016/j.ab.2010.05.014

15. www.apitherapy.com 16. www.apiservices.com 17. www.aca.org.ro/content/media/pagini/Mierea_pentru_Nutritie_si_Sanatate 18. www.proapicultura.ro 19. www.vforverde.ro/2014/03/cocktail-apicol-remediul-tuturor-afectiunilor 20. www.mierenaturalabio.ro/cosmetice-apicole.asp